CYTOCHEMISTRY: HISTORY, OBJECT OF STUDY, UTILITY AND TECHNIQUES - BIOLOGY - 2025

The cytochemistry comprises a series of techniques which rely on the identification and disposition of specific substances inside the cell. It is considered a branch of cell biology that combines cell morphology with chemical structure.

According to Bensley, founder of the application of modern cytology, expresses that the purpose of cytochemistry is to discover the chemical organization of cells in order to understand the mysteries of life. As well as studying the dynamic changes that occur during the different functional stages.

1: Paget's extramammary disease. (Hematoxylin-Eosin) 2: senile plaques observed in the cerebral cortex in a patient with Alzheimer's disease. (Silver impregnation) 3: Rabbit tongue, Collagen fibers (blue). Muscle fibers (purple strips). (Masson's trichrome). 4: Liver tissue with fatty degeneration. (Sudan III) 5: Inflamed liver. Necrosis. (Toluidine blue) Sources: Wikipedia. com / User: KGH / Public Domain files / Mohit Lalwani

In this way, it is possible to determine the metabolic role played by these substances within the cell.

Cytochemistry uses two main methods. The first is based on chemical and physical procedures. These techniques resort to the use of the microscope as an indispensable instrument to visualize the chemical reactions that occur on specific substances within the cell.

Example: the use of cytochemical dyes, such as the Feulgen reaction or PAS reaction, among others.

The second method is based on biochemistry and microchemistry. With this methodology it is possible to quantitatively determine the presence of intracellular chemicals.

Among the substances that can be revealed in a tissue or cell structure are the following: proteins, nucleic acids, polysaccharides and lipids.

History of cytochemistry

Cytochemical techniques since their invention have helped to understand the composition of cells, and over time a variety of techniques have emerged that use various types of dyes with dissimilar affinities and fundamentals.

Subsequently, cytochemistry opened new horizons with the use of certain substrates to show colorimetrically the presence of enzymes or other molecules within the cell.

Likewise, other techniques have emerged such as immunocytochemistry that has been of great help in the diagnosis of many diseases. Immunocytochemistry is based on antigen-antibody reactions.

On the other hand, cytochemistry has also used fluorescent substances called fluorochromes, which are excellent markers for the detection of certain cell structures. Due to the characteristics of fluorochrome, it highlights the structures to which it has been attached.

What do you study?

The various cytochemical techniques used on a biological sample have something in common: they reveal the presence of a specific type of substance and know its location within the biological structure under evaluation, be it a cell type or a tissue.

These substances can be enzymes, heavy metals, lipids, glycogen and defined chemical groups (aldehydes, tyrosine, etc.).

The information provided by these techniques can provide guidance not only for the identification of cells, but also for the diagnosis of various pathologies.

For example, cytochemical stains are very useful in differentiating between the various types of leukemias, since some cells express certain enzymes or key substances and others do not.

On the other hand, it should be noted that for the use of cytochemistry to be possible, the following considerations must be taken:

1) The substance must be immobilized in the place where it is naturally found.

2) The substance must be identified using substrates that react specifically with it and not with other compounds.

Utility

The samples that can be studied through cytochemical techniques are:

- Extended peripheral blood.

- Extended bone marrow.

- Tissues fixed for histochemical techniques.

- Cells fixed by cytocentrifugation.

Cytochemical techniques are highly supportive in the area of ​​hematology, as they are widely used to help in the diagnosis and differentiation of certain types of leukemias.

For example: Esterase reactions are used to differentiate myelomonocytic leukemia from acute monocytic leukemia.

The bone marrow and peripheral blood smears from these patients are similar, as some cells are difficult to identify morphologically alone. For this, the esterase test is carried out.

In the first, specific esterases are positive, while in the second, nonspecific esterases are positive.

They are also very useful in histology, since, for example, the use of the heavy metal staining technique (silver impregnation) stains the reticular fibers of an intense brown color in the myocardial tissue.

Techniques in cytochemistry

The most used techniques will be explained below:

- Use of colorants

The stains used are very diverse in cytochemical techniques and these can be classified according to several points of view:

According to the radical for which they have an affinity

They are divided into: acidic, basic or neutral. They are the simplest and the most used throughout history, allowing us to distinguish the basophilic components from the acidophilic ones. Example: hematoxylin-eosin staining.

In this case, the nuclei of the cells stain blue (they take hematoxylin, which is the basic dye) and the cytoplasms, red (they take eosin, which is the acid dye).

According to the color they provide

They can be orthochromatic or metachromatic. The orthochromatics are those that stain the structures of the same color that the dye has. For example, the case of eosin, whose color is red and stains red.

On the other hand, metachromatics stain structures a different color from theirs, such as toluidine, whose color is blue and yet stains violet.

Vital or supravital dyes

They are harmless dyes, that is, they color cells and they stay alive. These stains are called vital (eg trypan blue to stain macrophages) or supravital (eg Janus green to stain mitochondria or neutral red to stain lysosomes).

- Detection of lipids by means of fat-soluble dyes

Osmium tetroxide

It stains lipids (unsaturated fatty acids) black. This reaction can be observed with the light microscope, but because this dye is of high density it can also be visualized with an electron microscope.

Sudan III

It is one of the most used. This dye diffuses and solubilizes in the tissues, accumulating inside the lipid droplets. The color is scarlet red.

Sudan black stain B

It produces better contrast than the previous ones because it is able to dissolve also in phospholipids and cholesterol. It is useful for detecting azurophilic and specific granules of mature granulocytes and their precursors. Therefore it identifies myeloid leukemias.

- Aldehyde group staining (periodic acid Schiff stain)

Periodic acid Schiff stain can detect three types of aldehyde groups. They are:

- Free aldehydes, naturally present in tissues (plasma reaction).

- Aldehydes produced by selective oxidation (PAS reaction).

- Aldehydes generated by selective hydrolysis (Feulgen reaction).

PAS reaction

This staining is based on detecting certain types of carbohydrates, such as glycogen. Periodic acid Schiff breaks the CC bonds of carbohydrates due to the oxidation of glycolic groups 1-2, managing to liberate aldehyde groups.

Free aldehyde groups react with the Schiff's reagent and form a purple-red compound. The appearance of the purple-red color shows a positive reaction.

This test is positive in plant cells, detecting starch, cellulose, hemicellulose and peptins. While in animal cells it detects mucins, mucoproteins, hyaluronic acid and chitin.

In addition, it is useful in the diagnosis of lymphoblastic leukemias or erythroleukemia, among other pathologies of the myelodysplastic type.

In the case of acidic carbohydrates, the alcian blue stain can be used. The test is positive if a light blue / turquoise color is observed.

Plasmal reaction

The plasma reaction reveals the presence of certain long-chain aliphatic aldehydes such as palm and stearal. This technique is applied on frozen histological sections. It is treated directly with the Schiff's reagent.

Feulgen reaction

This technique detects the presence of DNA. The technique consists of subjecting the fixed tissue to a weak acid hydrolysis to later make it react with the Schiff's reagent.

Hydrolysis exposes the deoxyribose aldehyde groups at the deoxyribose-purine linkage. The Schiff's reagent then reacts with the aldehyde groups that were left free.

This reaction is positive in the nuclei and negative in the cytoplasms of the cells. Positivity is evidenced by the presence of a red color.

If this technique is combined with methyl green-pyronine, it is possible to simultaneously detect DNA and RNA.

- Cytochemical stains for protein structures

For this, the Millon reaction can be used, which uses mercury nitrate as a reagent. Structures containing aromatic amino acids will stain red.

- Cytochemical stains that use substrates to show the presence of enzymes

These stains are based on the incubation of the biological sample with a certain substrate and the product of the reaction subsequently reacts with diazo salts to form a colored complex.

Esterases

These enzymes are present in the lysosomes of some blood cells and are capable of hydrolyzing organic esters releasing naphthol. The latter forms an insoluble azo dye when it binds to a diazo salt, staining the site where the reaction occurs.

There are several substrates and depending on which one is used, specific esterases and nonspecific esterases can be identified. The former are present in immature cells of the myeloid series and the latter in cells of monocytic origin.

The substrate used for the determination of specific esterases is: naphthol-AS-D chloroacetate. While for the determination of non-specific esterases several substrates can be used such as naphthol AS-D acetate, alpha naphthyl acetate and alpha naphthyl butyrate.

In both cases the cells will stain deep red when the reaction is positive.

Myeloperoxidase

This enzyme is found in the azurophilic granules of granulocytic cells and monocytes.

Its detection is used to differentiate leukemias of myeloid origin from lymphoid ones. Cells containing myeloperoxidases turn ocher yellow.

Phosphatases

These enzymes release phosphoric acids from different substrates. They differ from each other according to the specificity of the substrate, the pH and the action of inhibitors and inactivators.

Among the best known are the phosphomonoesterases that hydrolyze simple esters (PO). Example: alkaline phosphatase and acid phosphatase, as well as phosphamidases that hydrolyze the bonds (PN). These are used to differentiate lymphoproliferative syndromes and for the diagnosis of hairy cell leukemia.

- Trichromic colorations

Mallary-Azan Trichrome

They are useful for differentiating the cytoplasm of cells from the fibers of connective tissue. Cells stain red and collagen fibers blue.

Masson's trichrome

This has the same utility as the previous one but, in this case, the cells stain red and the collagen fibers green.

- Dyes that stain specific organelles

Janus Green

It selectively stains the mitochondria.

Silver salts and osmic acid

Stains the Golgi apparatus.

Toluidine blue

Stains Nissi's bodies

Silver salts and PAS

They stain the reticular fibers and the basal lamina.

Orcein and fuchsin resorcin

They dye elastic fibers. With the first they are dyed brown and with the second deep blue or purple.

- Other techniques used in cytochemistry

Use of fluorescent substances or fluorochromes

There are techniques that use fluorescent substances to study the location of a structure in a cell. These reactions are visualized with a special microscope called fluorescence. Example: IFI technique (Indirect Immunofluorescence).

Detection of cellular components by immunocytochemistry

These techniques are very useful in medicine as they help to detect a certain cell structure and also quantify it. This reaction is based on an antigen-antibody reaction. For example: ELISA techniques (Enzyme Immuno Assay).

recommendations

- It is necessary to use control smears to evaluate the good performance of the dyes.

- Fresh smears should be used to undergo cytochemical staining. If not possible, they should be kept protected from light and stored at 4 ° C.

- Care must be taken that the fixative used does not negatively influence the substance to be investigated. In other words, it must be prevented from being able to extract or inhibit it.

- The time of use of the fixatives must be respected, since in general it should only last seconds, since exposing the smear for a longer time to the fixative can damage some enzymes.

References

1. "Cytochemistry." Wikipedia, The Free Encyclopedia. 30 Jun 2018, 17:34 UTC. 9 Jul 2019, 02:53 Available in: wikipedia.org
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3. Santana A, Lemes A, Bolaños B, Parra A, Martín M, Molero T. Cytochemistry of acid phosphatase: methodological considerations. Rev Diagn Biol. 200; 50 (2): 89-92. Available at: scielo.org
4. De Robertis E, De Robertis M. (1986). Cellular and molecular biology. 11th edition. Editorial Ateneo. Buenos Aires, Argentina.
5. Classic tools for studying cell biology. TP 1 (supplementary material) - Cell Biology. Available at: dbbe.fcen.uba.ar